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Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(3):656-665; doi:10.1093/carcin/bgn002
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice

Volker M. Arlt*, Marie Stiborová1, Colin J. Henderson2, Markus Thiemann3, Eva Frei4, Dagmar Aimová1, Rajinder Singh5, Gonçalo Gamboa da Costa6, Oliver J. Schmitz3, Peter B. Farmer5, C. Roland Wolf2 and David H. Phillips

Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
1 Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague 2, Czech Republic
2 Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Dundee DD1 9SY, UK
3 Department of Analytical Chemistry, University of Wuppertal, D-42119 Wuppertal, Germany
4 Division of Molecular Toxicology, German Cancer Research Center, D-69126 Heidelberg, Germany
5 Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, Leicester LE1 7RH, UK
6 Present address: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA

* To whom correspondence should be addressed. Tel: +44 208 722 4405; Fax: +44 208 722 4052; Email: volker.arlt{at}icr.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Many studies using mammalian cellular and subcellular systems have demonstrated that polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic versus extra-hepatic metabolism of BaP and its pharmacokinetics, we used the hepatic cytochrome P450 reductase null (HRN) mouse model, in which cytochrome P450 oxidoreductase, the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated intraperitoneally (i.p.) with 125 mg/kg body wt BaP daily for up to 5 days. Clearance of BaP from blood was analysed by high-performance liquid chromatography with fluorescence detection. DNA adduct levels were measured by 32P-post-labelling analysis with structural confirmation of the formation of 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene by liquid chromatography–tandem mass spectrometry analysis. Hepatic microsomes isolated from BaP-treated and untreated mice were also incubated with BaP and DNA in vitro. BaP–DNA adduct formation was up to 7-fold lower with the microsomes from HRN mice than with that from WT mice. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A. Pharmacokinetic analysis of BaP in blood revealed no significant differences between HRN and WT mice. BaP–DNA adduct levels were higher in the livers (up to 13-fold) and elevated in several extra-hepatic tissues of HRN mice (by 1.7- to 2.6-fold) relative to WT mice. These data reveal an apparent paradox, whereby hepatic CYP enzymes appear to be more important for detoxification of BaP in vivo, despite being involved in its metabolic activation in vitro.

Abbreviations: AhR, aryl hydrocarbon receptor; BaP, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; CYP, cytochrome P450; dG-N2-BPDE, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; EROD, 7-ethoxyresorufin O-deethylation; GST, glutathione S-transferase; HPLC, high-performance liquid chromatography; HRN, hepatic cytochrome P450 reductase null; i.p., intraperitoneally; LC–MS/MS, liquid chromatography–tandem mass spectrometry; NADH, reduced form of nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF, naphthoflavone; POR, cytochrome P450 oxidoreductase; PTGS, prostaglandin H synthase; SRM, selected reaction monitoring; TLC, thin-layer chromatography; WT, wild-type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The cytochrome P450s (CYPs) superfamily consists of a large number of haem-containing mono-oxygenases that play a pivotal role in the metabolism of many drugs and carcinogens (1). Much of the work carried out on the role of CYPs in xenobiotic metabolism has been done in vitro and has yielded vital information on CYP regulation and function. However, extrapolation from in vitro data to in vivo pharmacokinetics requires additional factors such as route of administration, absorption, renal clearance and tissue-specific CYP expression to be considered (2). A number of gene knockout and transgenic mice have been developed to study the role of specific enzymes in xenobiotic metabolism (3,4) and, although CYP knockout mouse models have provided important data on the effect of a single CYP enzyme on chemical-induced genotoxicity and carcinogenesis (5–10), the functional redundancy inevitably found in the CYP gene superfamily makes it difficult to determine the in vivo role of these enzymes in carcinogen metabolism as a whole (11). To overcome these limitations a mouse line, hepatic cytochrome P450 reductase null (HRN) has been developed in which cytochrome P450 oxidoreductase (POR), the unique electron donor to CYPs is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function (12). The HRN mouse can be used to establish the role of hepatic versus extra-hepatic xenobiotic metabolism and disposition (11,13).

Polycyclic aromatic hydrocarbons, of which benzo[a]pyrene (BaP) is the most commonly studied and measured, are formed by the incomplete combustion of organic matter (14). They are widely distributed in the environment and human exposure to them is unavoidable. Many compounds characterized as polycyclic aromatic hydrocarbons are found in tobacco smoke condensate, although for non-smokers, the principal route of exposure is through diet (15,16). A number of polycyclic aromatic hydrocarbons, including BaP, are mutagenic and carcinogenic, and they are widely believed to make a substantial contribution to the overall burden of cancer in humans (14,16,17). BaP requires metabolic activation prior to reaction with DNA, which is an essential step in the mechanism by which BaP exerts its genotoxic effects (18–20). The level of BaP–DNA adducts in cells is most probably the result of a balance between their formation and their loss through either DNA repair processes and/or apoptosis. Thus, BaP genotoxicity depends on various factors: (i) metabolic activation of BaP by phase I enzymes to reactive DNA-binding species; (ii) detoxification of reactive BaP metabolites by both phase I and phase II enzymes; (iii) rate of repair of BaP–DNA adducts and (iv) BaP-induced expression of genes such as those encoding enzymes involved in activation/detoxification or DNA damage response (10).

Based on evidence primarily from in vitro experiments, CYP1A1 and CYP1B1 are two of the most important enzymes in the metabolic activation of BaP, resulting in the formation of an epoxide that is then converted to a dihydrodiol by epoxide hydrolase (14,19). Further bioactivation by CYP1A1 and CYP1B1 leads to the formation of the ultimately reactive species, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) that can react with DNA, forming adducts preferentially at guanine residues, namely 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N2-BPDE) (20).

We observed recently that the carcinogen 3-nitrobenzanthrone, which is activated in vitro predominantly by cytosolic nitroreductases rather than microsomal POR (21), formed DNA adducts in tissues of HRN and wild-type (WT) mice at similar levels (21), whereas its metabolite 3-aminobenzanthrone, whose activation in vitro is primarily CYP dependent (22), formed significantly lower levels of adducts in the livers of HRN than of WT mice (23). Moreover, we found that in HRN and WT mice treated with the anti-neoplastic agent ellipticine, which forms DNA adducts mediated by CYPs and peroxidases, levels of hepatic ellipticine–DNA adducts were up to 65% lower in HRN mice, confirming the importance of CYPs in the bioactivation of ellipticine in the liver (24). In the present study we report the results of experiments on BaP activation by mouse hepatic microsomes and in various mouse organs using the HRN model, from which we draw contrasting conclusions on the role of CYPs in the genotoxicity of BaP in vivo and in vitro.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals
BaP (>96%) was purchased from Sigma–Aldrich (St Louis, MO). All other chemicals were of analytical purity or better.

Animal treatment
HRN (Porlox/lox + CreALB) mice on a C57BL/6 background (CXR Bioscience Ltd, Dundee, UK) used in this study were derived as described previously (12). Mice homozygous for loxP sites at the Por locus (Porlox/lox) were used as WT. BaP was dissolved in corn oil at a concentration of 12.5 mg/ml. Groups of female HRN and WT mice (3 months old, 25-30 g) were treated intraperitoneally (i.p.) with 125 mg/kg body wt (n = 3) of BaP daily either for 1 day (group I) or 5 days (group II). Control mice (n = 3) received corn oil only either for 1 day or for 5 days. Group I animals were killed 24 h after the single dose; group II animals were killed 24 h after the last dose. Several organs (liver, forestomach, glandular stomach, lung, colon, spleen, kidney and bladder) were removed, snap frozen and stored at –80°C until analysis.

Measurement of BaP–DNA adducts by 32P-post-labelling analysis
Genomic DNA from tissue was isolated by a standard phenol–chloroform extraction method and DNA adducts were measured for each DNA sample using the nuclease P1 enrichment version of the 32P-post-labelling method as described previously with minor modifications (25). Briefly, DNA samples (4 µg) were digested with micrococcal nuclease (120 mU, Sigma, Gillingham, UK) and calf spleen phosphodiesterase (40 mU, Calbiochem, Nottingham, UK), enriched and labelled as reported. Solvent conditions for the resolution of 32P-labelled adducts on polyethyleneimine–cellulose thin-layer chromatography (TLC) were as described (25,26). After chromatography, TLC plates were scanned using a Packard Instant Imager (Dowers Grove, IL) and DNA adduct levels (relative adduct labelling) were calculated from the adduct c.p.m., the specific activity of [{gamma}-32P]ATP and the amount of DNA (picomoles) used. Results were expressed as DNA adducts per 108 nucleotides. An external BPDE–DNA standard (27) was employed for identification of adducts in experimental samples. Separation of 32P-labelled 3',5'-deoxyribonucleoside bisphosphate adducts by high-performance liquid chromatography (HPLC) was done as described recently (28). Briefly, individual adduct spots detected by the 32P-post-labelling TLC assay were excised from the TLC plates, extracted and co-chromatographed with standard bisphosphate adduct as reported. Radioactivity eluting from the reversed-phase column was measured by monitoring Cerenkov radiation with a Flow Scintillation Analyzer (Packard).

Identification of BaP–DNA adducts by liquid chromatography–tandem mass spectrometry
A stable-isotope internal standard of dG[15N5]-N2-BPDE was prepared as described (29). The formation of dG-N2-BPDE was analysed in DNA samples (50 µg) using positive electrospray ionization liquid chromatography–tandem mass spectrometry (LC–MS/MS) with selected reaction monitoring (SRM) (29). For liver DNA, samples from individual animals were analysed, whereas for the remaining tissues, DNA samples were pooled from three animals.

Detection of BaP in blood by HPLC analysis
On day 1 sequential blood samples (10 µl) were obtained by tail-bleed at 0.5, 1, 1.5, 2, 4, 6, 8 and 12 h after injection of BaP. Blood samples were stored at –80°C until analysis. Frozen blood samples were thawed at room temperature and 15 µl of a solution of benzo[e]pyrene (Dr. Ehrensdorfer GmbH, Augsburg, Germany) in acetonitrile (1 µg/ml) was added as internal standard. Whole blood was extracted three times with 100 µl ethyl acetate:acetone (2:1, vol:vol). Organic extracts were pooled and dried for ~2 h in a vacuum centrifuge. The residue was resuspended in acetonitrile (150 µl) using an ultrasonic bath for 5 min. Each sample, alternating with the control sample, was analysed three times by HPLC (Merck Hitachi, Darmstadt, Germany) with a fluorescence detector. Injection volume was 20 µl, and samples were separated on a LiChroCART reversed-phase column [Merck LiChroCART 250-4, RP-18 (5 µm)]. The mobile phase was acetonitrile:water (85:15, vol:vol) at a flow rate of 1 ml/min. Fluorescence excitation and emission wavelengths were 294 and 404 nm, respectively. BaP concentrations in blood were calculated by comparing the peak area of BaP with those of benzo[e]pyrene. The response factor of BaP compared with benzo[e]pyrene was determined as 10.

Pharmacokinetic analysis
Pharmacokinetic variables were calculated with WinNonLin software version 5.1 using the non-compartmental model 200 for extravascular input: AUC, area under the curve; t1/2 terminal half-life; Cmax, maximal blood concentration; Tmax, time of Cmax; Cl, clearance. Differences in pharmacokinetic parameters between HRN and WT mice were analysed by the Mann–Whitney test and considered significant at P < 0.05.

Preparation of microsomes
Hepatic microsomes from HRN and WT mice (group II), untreated or treated with BaP for 5 days as described above, were isolated as described previously (30). Pooled microsomal fractions were used for further analyses.

Preparation of CYP1A1 and POR antibodies
Recombinant rat CYP1A1 protein was purified to homogeneity from membranes of Escherichia coli transfected with a modified CYP1A1 cDNA (31). Rabbit liver POR was purified as described (32). Leghorn chickens were immunized subcutaneously three times (with 1 week interval) with rat recombinant CYP1A1 and rabbit hepatic POR antigens (0.1 mg per animal) emulsified in complete Freund’s adjuvant for the first injection and in incomplete adjuvant for boosters. Immunoglobulin fraction was purified from pooled egg yolks using fractionation by polyethylene glycol 6000 (31). Mice anti-human prostaglandin H synthase (PTGS) 1 and 2 (PTGS1 and PTGS2) antibodies were obtained from Gentest Corp. (Woburn, MA).

Determination of CYP1A, POR and PTGS protein levels in hepatic microsomes
Immunoquantitation of hepatic microsomal CYP1A1 and CYP1A2, POR, PTGS1 and PTGS2 was done essentially as described previously using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (31,33). CYP, POR and PTGS were probed with the chicken anti-rat CYP1A1, chicken anti-rabbit POR and mouse anti-human PTGS polyclonal antibodies as reported (31,33). The antibody against rat recombinant CYP1A1 recognizes both CYP1A1 and CYP1A2 in mice liver microsomes. Rat recombinant CYP1A1 and CYP1A2 (in SupersomesTM, Gentest Corp.), rabbit POR, ovine PTGS1 and human recombinant PTGS2 (Gentest Corp) were used as positive controls to identify protein bands in microsomal samples. The antigen–antibody complex was visualized with an alkaline phosphatase-conjugated rabbit anti-chicken IgG antibody and 5-bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium as chromogenic substrate (31,33). The detection limit was 0.005 pmol CYP1A per lane (31,33) and 0.01 pmol for the other enzymes.

Determination of CYP1A and POR enzymatic activity in hepatic microsomes
The hepatic microsomal samples were characterized for CYP1A activity using 7-ethoxyresorufin O-deethylation (EROD) activity (31,33). The activity of POR was measured as reported previously (34).

Real-time quantitative polymerase chain reaction
Total RNA was isolated from livers of HRN and WT mice killed after 5 days (either untreated or treated with BaP; group II). Tissue (~20 mg) was homogenized in 600 µl trizol (Invitrogen, Paisley, UK). After addition of chloroform (120 µl), samples were centrifuged for 20 min at >10 000g (4°C), and the supernatant was mixed with 70% ethanol (600 µl). Total RNA was extracted using the Qiagen RNeasy Mini Kit protocol (RNeasy Mini Handbook, Qiagen, Crawley, UK) as described (35). Two-step reverse transcription–polymerase chain reaction was used to generate cDNA for relative quantitation analysis using real-time fluorescent polymerase chain reaction on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Warrington, UK) performed as previously described (35). To detect the modulated expression of selected target genes 20x Assays-On-DemandTM gene expression primers and probes (Applied Biosystems) were used (PTGS1-Mm00477214_m1, PTGS2-Mm00478374_m1 and MPO-Mm00447885_m1). All polymerase chain reactions were performed in triplicate and relative gene expression was calculated using the comparative threshold cycle (CT) method as performed previously (35).

Microsomal incubations
Incubation mixtures consisted of 50 mM potassium phosphate buffer (pH 7.4), 1 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced form of nicotinamide adenine dinucleotide (NADH), pooled hepatic microsomal fraction (0.5 mg protein) from HRN and WT mice (either untreated or treated with BaP; group II), 0.1 mM BaP (dissolved in 7.5 µl dimethyl sulphoxide) and calf thymus DNA (0.5 mg) in a final volume of 750 µl. Incubations were also carried out in the presence of a PTGS cofactor, arachidonic acid (36,37). Mixtures then contained 0.1 mM arachidonic acid as cofactor instead of NADPH, and additionally 5 mM magnesium chloride. Incubations were carried out at 37°C for 90 min; the micosomal-mediated BaP–DNA adduct formation was linear up to 120 min. Control incubations were carried out (i) without microsomes; (ii) without NADPH or arachidonic acid; (iii) without DNA and (iv) without BaP. After the incubation, DNA was isolated by a standard phenol–chloroform extraction method.

Inhibition studies
The following chemicals were used to inhibit the activation of BaP in mouse hepatic microsomes: {alpha}-naphthoflavone ({alpha}-NF), which inhibits CYP1A1 and CYP1A2 (22,37); ellipticine, which competes with CYP1A1 substrates, thus inhibiting efficiently CYP1A1-mediated oxidation of other substrates (30,38); indomethacin, a selective inhibitor of PTGS (37); and {alpha}-lipoic acid, which inhibits POR (34). Inhibitors were dissolved in 7.5 µl of methanol, to yield final concentrations of 0.1 mM in the incubation mixtures. Mixtures were then incubated at 37°C for 10 min with NADPH prior to adding BaP, and then incubated for a further 90 min at 37°C. After the incubation, DNA was isolated as mentioned above.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
DNA adduct formation in mice
DNA adduct formation in organs (liver, lung, forestomach, glandular stomach, kidney, bladder, spleen and colon) of HRN and WT mice treated i.p. with either a single dose or five daily doses of BaP was analysed by 32P-post-labelling. On TLC the DNA adduct pattern consisted of a single spot with all organs from BaP-treated animals (Figure 1A and B, inset), whereas no DNA adducts were detected in control animals (data not shown). The identity of the material in the adduct spots was determined by HPLC to be the reactive metabolite BPDE bound to the N2 position of guanine (dG-N2-BPDE) both in HRN and in WT mice (Figure 1C) (27). Independent corroboration of the structure of the DNA adducts in all eight tissues examined was obtained by LC–MS/MS SRM (29), using a stable-isotope internal standard of dG[15N5]-N2-BPDE; representative results for liver only are shown in Figure 2.


Figure 1
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  		Fig. 1. Autoradiographic profiles of BaP-derived DNA adducts obtained by 32P-post-labelling. Adduct spots obtained after TLC 32P-post-labelling using nuclease P1 enrichment (inset) were excised and extracted from the plates, dissolved and injected on HPLC. DNA adduct pattern obtained in liver DNA of (A) HRN and (B) WT mice treated (i.p.) with 125 mg/kg body wt BaP once daily for 5 days. These profiles are representative of adduct profiles obtained with DNA from other mouse tissues, including lung, forestomach, glandular stomach, kidney, bladder, spleen and colon. (C) DNA adduct pattern obtained in salmon testis DNA modified with BPDE in vitro (27). The arrow indicates the position of the 5'-32P-labelled bisphosphate dG-N2-BPDE adduct.

 


Figure 2
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  		Fig. 2. Determination of dG-N2-BPDE adducts by LC–MS/MS SRM in mouse liver DNA of (A) HRN and (B) WT mice treated (i.p.) with 125 mg BaP/kg body wt daily for 5 days (5d). These profiles are representative of adduct profiles obtained with DNA from other mouse tissues including lung, forestomach, glandular stomach, kidney, bladder, spleen and colon. Typical LC–MS/MS SRM ion chromatograms are shown for the transition m/z 570 to 454 and m/z 575 to 459 for dG-N2-BPDE and dG[15N5]-N2-BPDE, respectively. Peaks I and II represent stereoisomers of the dG-N2-BPDE adduct, peak II corresponds to the stereoisomer with the 7R,8S,9S-trihydroxy-trans-(10S)-dG[15N5]-N2-BPDE configuration. After 1 day (group I; mean ± SD; n = 3) the adduct levels in liver were 1916.2 ± 130.7 and 116.3 ± 36.6 dG-N2-BPDE adducts per 108 nucleotides in HRN and WT mice, respectively. After 5 days (group II; mean ± SD; n = 3) adduct levels in liver were 3115.9 ± 551.3 and 264.9 ± 39.1 dG-N2-BPDE adducts per 108 nucleotides in HRN and WT mice, respectively.

 
DNA adduct levels were quantitated by both 32P-post-labelling and LC–MS/MS. Figure 3 shows the results of 32P-post-labelling, in which it can be seen that after a single dose of BaP (Figure 3A), significantly higher levels of adducts (up to 2.6-fold) were formed in 5/7 of the extra-hepatic tissues (P < 0.05) of the HRN mice that lack hepatic CYP activity. This difference was less evident after mice had received multiple doses of BaP (Figure 3B), when only two of the extra-hepatic tissues of HRN mice, lung and colon, had significantly more adducts than were found in WT mice. The greatest differences between HRN and WT mice, however, were found with liver DNA adducts. After a single dose of BaP, adduct levels were 13.4-fold higher in HRN mice (Figure 3A), and the difference was 6.4-fold after multiple doses (Figure 3B) (P < 0.01). Quantitation of hepatic dG-N2-BPDE levels by LC–MS/MS SRM revealed that DNA binding in HRN mice was up to 16.5-fold higher than that in WT mice (see legend Figure 2). In the present study, DNA adduct levels determined by LC–MS/MS SRM were generally 2- to 3-fold higher than those determined by TLC 32P-post-labelling, an observation which is consistent with previous results (29).


Figure 3
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  		Fig. 3. Quantitative TLC32P-post-labelling analysis of dG-N2-BPDE adducts in organs of HRN and WT mice treated (i.p.) (A) once (1d) or (B) daily with 125 mg BaP/kg body wt for 5 days (5d). F, fold increase in DNA binding in HRN mice compared with WT mice. Values are given as mean ± SD (n = 3); each DNA sample was determined by two post-labelled analyses. Comparison was performed by t-test analysis: *P < 0.01, #P < 0.05, different from WT. RAL, relative adduct labelling.

 
BaP clearance in blood
The concentration of BaP in blood after i.p. treatment of HRN and WT mice with a single dose of BaP was determined using HPLC with fluorescence detection (Figure 4). Pharmacokinetic analysis revealed no statistically significant difference in any of the analysed parameters (e.g. clearance, terminal half-life and area under the curve). As can be seen in Figure 4 the Cmax was slightly higher (~1.25-fold) in HRN relative to WT mice, but this difference was not statistically significant.


Figure 4
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  		Fig. 4. Comparison of BaP clearance in blood between HRN and WT mice treated once with 125 mg BaP/kg body wt. Values are given as mean ± SD (n = 3). Inset: pharmacokinetic variables in HRN and WT mice calculated with non-compartmental analysis.

 
The effect of BaP on expression of hepatic biotransforming enzymes
Using western blot analysis with polyclonal antibodies raised against CYP1A1, POR or PTGS, the protein expression levels of these enzymes were determined in hepatic microsomes isolated from HRN and WT mice from group II. Both CYP1A enzymes are constitutively expressed in livers of untreated HRN and WT mice, with HRN mice having marginally higher levels (1.4-fold) than WT mice (Figure 5A). Treatment with BaP led to 98-fold higher expression levels of CYP1A in livers of WT mice, but induced its levels 175-fold in HRN mice. The increase in CYP1A levels in these WT mice was associated with a strong increase in EROD activity, a measure of CYP1A enzyme activity (Figure 5C). EROD activity was also observed in HRN mice treated with BaP, but it was 3.4-fold lower than that in WT mice treated with BaP, whereas no EROD activity was detectable in untreated HRN mice.


Figure 5
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  		Fig. 5. Expression of (A) CYP1A and (B) POR and their enzymatic activity [EROD (C) and POR activity (D)] in livers of HRN and WT mice, control (untreated) mice or mice treated (i.p.) with 125 mg BaP/kg body wt daily for 5 days. Inset in A and B: immunoblots of microsomal CYP1A and POR from each mouse group, stained with antibody against rat CYP1A1 and rabbit POR, respectively. Pooled hepatic microsomal samples were used for analyses as described in Material and methods. Values are given as mean ± SD (n = 3). ND, not detectable.

 
Hepatic POR expression was detected in WT mice, while as expected, its levels in HRN mice were very low, but still detectable, by immunostaining (Figure 5B). Hepatic POR levels in HRN mice were estimated to be <1.5% of the levels in WT mice (Figure 5B). POR activity measured with cytochrome c as a substrate was, however, not detectable in hepatic microsomes of HRN mice (Figure 5D). Surprisingly, the expression level of POR was also slightly induced in hepatic microsomes from mice treated with BaP (Figure 5B), where we found a 1.6- and 2.9-fold increase in WT and HRN mice, respectively. It is noteworthy that POR activity was detectable in HRN mice treated with BaP (Figure 5D). However, no protein expression of PTGS1 and PTGS2 was detectable in hepatic microsomes under any of the experimental conditions used (data not shown).

Besides the evaluation of the effects of BaP on hepatic CYP-related enzymes, modulation of PTGS and myeloperoxidase mRNA expression by BaP in these mice was also investigated. Total RNA was isolated from frozen livers of group II mice and the relative amounts of PTGS1, PTGS2 and MPO mRNAs were measured by real-time polymerase chain reaction. No expression of PTGS2 and MPO was observed and no BaP-induced change in expression of PTGS1 mRNA was detectable under the experimental conditions used (data not shown). No differences in PTGS1 mRNA levels were observed between WT and HRN mice (data not shown).

Activation of BaP by hepatic microsomes
We determined DNA adduct formation by BaP in in vitro incubations with calf thymus DNA in the presence of microsomes isolated from livers of HRN and WT mice from group II. Hepatic microsomes from both mice strains were capable of activating BaP to form DNA adducts (Figure 6). Using TLC 32P-post-labelling, one major DNA adduct spot was detected, corresponding to the one found in all the BaP-treated mouse tissues (compare Figure 1), and verified by co-chromatography analyses on HPLC as being dG-N2-BPDE (data not shown). Selected in vitro samples were analysed by LC–MS/MS to examine the formation of dG-N2-BPDE. Overall, the levels of this DNA adduct determined by LC–MS/MS were 2- to 5-fold higher than those determined by TLC 32P-post-labelling (data not shown). Hence, these findings are consistent with the results obtained for the in vivo samples (see above).


Figure 6
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  		Fig. 6. DNA adduct formation by BaP activated with microsomes isolated from livers of HRN and WT mice, control mice (untreated) (A) or mice treated (i.p.) with 125 mg BaP/kg body wt daily for 5 days (B) as determined by TLC 32P-post-labelling. F, fold increase in DNA binding in microsomes from WT mice compared with HRN mice. Values are given as mean ± SD (n = 2); each DNA sample was determined by two post-labelled analyses. RAL, relative adduct labelling. AA, arachidonic acid; Control, microsomes and DNA only; E, ellipticine; IM, indomethacin; {alpha}-LA, {alpha}-lipoic acid; . ND, not detected.

 
As shown in Figure 6, the formation of BaP–DNA adducts was stimulated by NADPH and NADH. DNA adduct formation catalysed by hepatic microsomes from mice pretreated with BaP was generally higher (Figure 6B) than that with hepatic microsomes isolated from untreated mice (Figure 6A). NADPH, a cofactor for CYP-dependent oxidation of BaP, was the most effective cofactor in both systems. DNA adduct levels were up to 7-fold higher after activation with hepatic microsomes isolated from livers of WT than those from HRN mice. After the addition of NADH, which is a cofactor of the microsomal NADH:cytochrome b5 oxidoreductase, a second electron donor for CYP-dependent systems (39), BaP–DNA adduct formation was up to 2.7-fold higher with hepatic microsomes from WT than with microsomes from HRN mice. Inhibition experiments supported the role of CYPs in the activation of BaP by hepatic microsomes. {alpha}-Lipoic acid, a selective inhibitor of POR (34), reduced DNA adduct formation by 70–90%. To further investigate the role of hepatic CYP1A1 and CYP1A2 enzymes in BaP activation, incubations were carried out in the absence and presence of {alpha}-NF, a specific inhibitor of CYP1A1 and CYP1A2 (22,30), and ellipticine, which is utilized as an inhibitor of CYP1A1 (30,38). Both compounds inhibited DNA binding by 70–90%, suggesting that most of the hepatic microsomal activation of BaP in vitro is attributable to CYP1A1 enzyme activity in both mouse strains. It is noteworthy that although no DNA adducts were observed in control incubations with hepatic microsomes isolated from untreated mice, DNA adduct formation was detected in control incubations with hepatic microsomes isolated from BaP-treated mice, even without the addition of BaP to these incubations (Figure 6B).

We also considered other BaP-activating enzymes such as PTGS. Arachidonic acid, a cofactor for PTGS-dependent oxidation (23,37), did not influence BaP–DNA adduct formation by microsomes from either mouse strain, with or without BaP pretreatment (Figure 6). However, indomethacin, a selective inhibitor of PTGS (37), inhibited BaP activation in incubations with hepatic microsomes isolated from BaP-treated HRN mice by 30–40% (Figure 6B), although no such inhibition of the very low levels was obtained with microsomes isolated from BaP-treated WT mice (Figure 6B).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
CYP1A1 is believed to be one of the major enzymes responsible for the metabolic activation of BaP in organisms (14,19). CYPs comprise a large family of subfamilies and isoenzymes showing considerable redundancy and overlapping substrate specificity (1). To examine the effect of CYPs as a whole on hepatic BaP metabolism and metabolic activation, we utilized the HRN mouse (12), which carries a deletion of the POR gene specifically in the liver and thus lacks CYP function in hepatocytes (11). A limited number of treatments of mice with the dose used in this study, 125 mg/kg body wt, is carcinogenic (40) and induces mutagenicity in multiple organs (41).

In the present study, we showed the specific role of hepatic CYP enzymes in the activating pathways of BaP in vitro. BaP–DNA adduct formation was strongly dependent on the catalytic activities of POR present in the mouse hepatic microsomes assayed. This conclusion was further supported by the inhibition of BaP–DNA adduct formation with {alpha}-lipoic acid, a specific inhibitor of POR (34). Using specific CYP inhibitors, namely {alpha}-NF and ellipticine (33,38), most of the CYP-mediated BaP activation was attributable to CYP1A activity. This is in line with recent results from our laboratory showing that in mouse hepatoma Hepa1c1c7 cells BaP–DNA adduct formation correlated with induction of CYP1A1 (26). Utilization of hepatic microsomes isolated from mice pretreated with BaP to activate BaP in vitro demonstrated that DNA adduct levels were significantly higher than those obtained with hepatic microsomes isolated from untreated mice. DNA binding by BaP catalysed by hepatic microsomes isolated from treated WT mice was much higher than that obtained with microsomes isolated from treated HRN mice. Since hepatic CYP enzyme activity had been essentially obliterated by the conditional deletion of POR in hepatocytes, the level of BaP activation to DNA adducts in microsomes isolated from HRN mice is difficult to rationalize. One possible explanation is that endoplasmic membranes from non-parenchymal cells that still contain POR are mixed with those from hepatocytes containing CYP in the process of microsome isolation. This, in combination with the finding that BaP is a strong CYP inducer maybe also in otherwise not very active non-parenchymal cells may explain these in vitro findings. The increase in DNA adduct formation upon pretreatment with BaP catalysed by microsomes from WT mice is only 28.3-fold, whereas it is 50.5-fold in HRN mice, indicating that another BaP-inducible activating mechanism, independent of POR, is responsible for BaP activation in vitro in these mice. Similarly, residual in vitro metabolism of the anticancer drug cyclophosphamide was observed in incubations using hepatic microsomes isolated from HRN mice (13). Moreover, a recent study showed that pulmonary CYPs play a major role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butone-induced lung tumours and that the number of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butone-induced lung tumours were reduced in pulmonary POR-null mice but were increased in hepatic POR-null mice relative to WT control (42). However, lung tumours in pulmonary POR-null mice were positive for POR expression because POR deletion occurred in only a portion of the Clara cells and the alveolar type II epithelial cells, indicating that the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butone-induced tumours were derived from POR-expressing alveolar type II epithelial cells.

Another explanation could be the induction of metabolizing enzymes other than CYPs. In hepatic microsomes isolated from BaP-treated HRN mice, DNA adduct formation was observed even without the addition of BaP, suggesting that BaP and/or its metabolites can bind to hepatic microsomal membranes, but it is subsequently released to react with DNA during the in vitro incubation. The latter effect was much more pronounced in hepatic microsomes isolated from HRN mice, suggesting the induction of metabolizing enzymes in the latter microsomes. BaP is not only a good substrate for CYP1A1 and CYP1B1, but also for PTGS2 (43). When we used arachidonic acid, a cofactor for PTGS2, in hepatic microsomes isolated from mice treated with BaP, no increase in BaP–DNA adduct formation was observed. This is consistent with no detectable PTGS proteins in hepatic microsomes of either mouse strain.

Previous studies demonstrated that for a genotoxin activated by a CYP-independent mechanism, 3-nitrobenzanthrone, DNA adduct formation in mouse tissues was the same in HRN and WT mice (21), whereas DNA binding by another genotoxin, 3-aminobenzathrone, which is metabolically activated by CYPs, was elevated in the extra-hepatic tissues and reduced in the livers of HRN mice (23). If CYP1A1 in particular, or CYPs in general, are involved in the activation of BaP in vivo, it would be predicted that there would be elevated levels of BaP–DNA adducts in extra-hepatic tissues and reduced levels in the liver, of HRN mice, relative to WT mice. While we did indeed detect more adducts in extra-hepatic tissues, we also found that the levels in liver were vastly elevated, not reduced.

Our studies on the consequences of organ-specific loss of all CYP function bear comparison with other studies on the systemic loss of function, or abrogation of induction, of specific CYPs. Administration of a high dose of BaP i.p. to Cyp1a1(–/–) mice resulted in slower clearance and higher adduct levels (4-fold) in liver than those in Cyp1a1(+/–) mice (44). Repeated high doses were less toxic to the homozygous knockout mice than to the heterozygotes. The results were interpreted to indicate that absence of CYP1A1 protects the intact animal from BaP-mediated liver toxicity by decreasing the formation of large amounts of toxic metabolites, while the slower metabolic clearance of BaP resulted in greater formation of BaP–DNA adducts in the livers of the Cyp1a1(–/–) mice (44). Conversely, BaP was more toxic to Cyp1a1(–/–) mice than to Cyp1a1(+/+) WT mice given by oral administration (10). The rate of clearance was still slower (4-fold) in the knockout mice, and higher levels of DNA adducts were observed in liver, spleen and bone marrow, but not in small intestine, accompanied by qualitative differences in the DNA adduct patterns between the two genotypes. Combined studies with Cyp1a1(–/–) and Cyp1b1(–/–) single knockout and Cyp1a1/1b1(–/–) double knockout mice administrated BaP orally essentially mirrored these findings (45). In the present study, we chose i.p. injection as the administration route to achieve a high induction of hepatic BaP metabolism. However, although we found much higher DNA adduct levels in the livers of HRN mice than WT mice, we found no statistically significant difference in BaP pharmacokinetics (e.g. clearance, terminal half-life and area under the curve) in HRN relative to WT mice.

BaP induces CYP1A1 through binding to the aryl hydrocarbon receptor (AhR), a transcription factor that also regulates the expression of a number of other phase I and phase II xenobiotic metabolizing enzyme genes. Mice that lack AhR have been found to be refractory to BaP carcinogenesis after the compound was administered topically or subcutaneously, a finding that was interpreted as demonstrating that AhR is involved in BaP carcinogenesis and in converting BaP to its active metabolites (46). However, when BaP was administered i.p. to AhR(–/–) mice, total BaP–DNA adduct formation was at similar levels as in WT mice, although the levels of individual post-labelling adduct spots varied (47). In another study in which BaP was given by gavage to AhR(–/–) mice, liver adduct levels were significantly higher than those in the WT mice (48). The authors observed slower clearance of BaP in the knockout mice, as was also observed by Uno et al. (10) in the Cyp1a1(–/–) mice and suggested that this may explain, at least partly, the increase in DNA adduct levels. However, our present study with HRN mice did not indicate slower clearance of BaP, thereby excluding such an explanation for the high liver DNA adducts that we detected, if it can be assumed that concentrations of BaP in blood reflect tissue concentrations. The formation of BaP–DNA adducts in incubations of hepatic microsomes from BaP-pretreated mice, without the addition of BaP, suggests some BaP accumulated in the livers of the HRN mice. While the in vivo studies together represent a variety of doses, durations of treatment and routes of administration, overall the impression is that the function of hepatic CYPs is to detoxify BaP, not to activate it, which is in direct contrast to the general assumption from in vitro studies which point to metabolic activation of BaP by this enzyme system.

The mechanism by which DNA-binding species were generated in liver from BaP in the present study is not known, but it is clear that the process did not involve generation of a different reactive species from that formed in WT mice. The DNA adduct patterns obtained by 32P-post-labelling were the same for the knockout and WT mice for liver and, indeed, for extra-hepatic tissues, and the unequivocal identification of the major adduct as being the product of the reaction of BPDE with guanine residues in DNA was confirmed by MS. Previously, a very good correlation was obtained between BaP–DNA adduct determination by 32P-post-labelling and LC–MS/MS SRM (29). If, as is suggested by our study and those of other investigators (47,48), metabolic activation of BaP in vivo is mediated by CYP-independent and/or AhR-independent pathways, it is apparent from our own studies, if not from others, that the end result, i.e. the nature of the DNA adducts formed, is the same. Besides differences in CYP enzyme activities the above-mentioned PTGS2 may be of importance to explain the high BaP–DNA adduct levels found in livers of HRN mice, which lack almost all POR-mediated CYP-catalysed bioactivation. However, we did not find any differences neither in PTGS2 protein levels nor PTGS2 gene expression. Therefore, the role of other, as-yet-unidentified, BaP-activating enzymes awaits further investigation.

It is noteworthy that microarray gene expression technology previously showed that the absence of POR in the liver, and thus hepatic CYP function, appears to result only in minor compensatory changes in the expression of genes encoding for phase I and phase II xenobiotic metabolizing enzymes in untreated animals (49). Among them only the glutathione S-transferases (GSTs), namely GSTM2 and GSTM3, showed significant induction. Diol epoxides such as BPDE are detoxified by GSTs, in particular GSTM1 (19), and the ratio between CYP1A1 and GST enzyme activities has been described as a critical determinant of the target dose of reactive DNA-binding BPDE (50). As epidemiological studies indicate a dependence of BaP–DNA adduct levels on the CYP1A1 and GSTM1 genotype (50), the expression of GSTs may also need consideration to explain the increased BaP-induced DNA adduct formation in HRN mice.

Numerous studies have shown that BaP is metabolized by CYPs to phenols, quinones, epoxides and arene oxides (19). In mouse hepatoma Hepa1c1c7 cells, which express inducible CYP1A1, 3-hydroxybenzo[a]pyrene was the major metabolite formed after exposure to BaP (26) and this BaP metabolite is known to inhibit mutagenesis and tumorigenesis (51). Thus, although the metabolites of BaP were not determined in the present study, it is tempting to speculate that increased CYP-catalysed formation of 3-hydroxybenzo[a]pyrene in WT mice may inhibit BaP-induced DNA adduct formation to a greater extent than HRN mice. This would be the case especially after BaP-induced CYP activity, which was much higher in livers of WT mice than HRN mice.

In general, i.p. administration results in the uptake of BaP by mesenteric veins and lymphatic system that go directly to the liver, bypassing the gastrointestinal tract. It was pointed out previously that important pharmacokinetic differences depend on the route of administration (10). After oral administration, BaP uptake would be via the gastrointestinal tract and then to the liver. Therefore, it would be interesting to dissect the first-pass elimination further by using a gastrointestinal tract- and liver-specific conditional POR knockout mouse line. Recently, a variant on the HRN mouse line (Porlox/lox/CreCYP1A1) has been developed in which POR can be deleted conditionally in the liver and gastrointestinal tract using the rat CYP1A1 promoter to drive Cre recombinase expression (52). Using this line, administration of the CYP1A1 inducers tetrachlorodibenzo-p-dioxin or β-NF results in both hepatic and gastrointestinal deletion of POR, whereas administration of 3-methylcholanthrene results specifically in the loss of hepatic POR expression. In future, we will test BaP in this new line, which potentially provides an improved model to investigate the tissue-specific balance between BaP activation and detoxification.

In summary we have demonstrated that in vivo hepatic CYPs are more important for BaP detoxification than activation. Our results reveal an apparent paradox as CYPs mediate the metabolic activation of BaP in vitro. However, our study may also point towards the presence of other, as-yet-unidentified, hepatic BaP-activating enzymes in addition to PTGS2. Various studies, including ours, clearly show that expression and induction of CYPs can be beneficial in carcinogen metabolism because they may detoxify the noxious agent. The HRN mouse provides an excellent model for further investigation of these effects.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Cancer Research UK (to C.R.W. and D.H.P.); Grant Agency (203/06/0329 to M.S.); Ministry of Education of the Czech Republic (MSM0021620808 to M.S.); German Research Foundation (to O.J.S.); UK Medical Research Council (G0100873 to P.B.F.); Environmental Cancer Risk, Nutrition and Individual Susceptibility; European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (513943 to V.M.A., C.J.H., R.S., G.G.d.C., P.B.F., C.R.W. and D.H.P.).


   Acknowledgments
 
We thank Florence Raynaud, Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK, for assistance with the pharmacokinetic analysis.

Conflict of Interest Statement: The HRN mouse is a trademark of CXR Biosciences Ltd, Dundee Technopole, James Lindsay Place, Dundee, UK. C.R.W. is a Director, cofounder and shareholder and C.J.H. is a consultant to CXR Biosciences Ltd.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received August 22, 2007; revised December 11, 2007; accepted December 27, 2007.


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